A series of defenses against oxidative stress by oxygen that causes disease processes involves well-regulated enzyme reactions catalyzed by superoxide dismutases (SOD), catalase, peroxidases, and reductases. Interruptions in these reactions, such as ischemia and reperfusion, have been known to cause irreversible damage in cells and tissues. In some diseases involving genetic disorders, i.e. Down's syndrome, overexpression of enzymes such as Cu,Zn-SOD is implicated as a probable cause. We investigated the effects from enhancement of SOD activity and searched for the damaging species that might produce these effects. Electron paramagnetic resonance (EPR) spectroscopy and a spin trapping method were used for the studies. The SOD catalyzes the reaction of superoxide dismutation and thus protects biomolecules from the superoxide toxicity. However, its reaction product, hydrogen peroxide, is also toxic and is known to inactivate Cu,Zn-SOD itself. In this study we found that during the inactivation of Cu,Zn-SOD by hydrogen peroxide, "free" hydroxyl radicals are produced and escape from the active-site channel. We also found that, in the presence of anionic radical scavengers, the enzyme is protected against the inactivation by hydrogen peroxide and continues producing radicals originating from the scavengers. In this case Cu,Zn-SOD behaves as an enzyme that catalyzes the formation of radicals of corresponding scavengers using hydrogen peroxide as a cofactor. One scavenger with this property is glutamate, a neurotransmitter. The finding that Cu,Zn-SOD is capable of catalyzing the formation of hydroxyl radicals and scavenger radicals may in part explain observations that elevated intracellular SOD activity causes more harmful effects to cells. The scavenger radicals that might have less reactivity can travel farther and may exert more specific damage in vivo.